In Drosophila melanogaster the COM Locus Directs the Somatic Silencing of Two Retrotransposons through both Piwi-Dependent and -Independent Pathways

BACKGROUND: In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements. This RNA silencing involves small RNAs of 26-30 nucleotides that are mainly produced from the antisense strand and function through the Piwi protein. Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus. In addition to this germ-line expression, Piwi has also been implicated in diverse functions in somatic cells. PRINCIPAL FINDINGS: Here, we show that two LTR retrotransposons from Drosophila melanogaster, ZAM and Idefix, are silenced by an RNA silencing pathway that has characteristics of the rasiRNA pathway and that specifically recognizes and destroys the sense-strand RNAs of the retrotransposons. This silencing depends on Piwi in the follicle cells surrounding the oocyte. Interestingly, this silencing is active in all the somatic tissues examined from embryos to adult flies. In these somatic cells, while the silencing still involves the strict recognition of sense-strand transcripts, it displays the marked difference of being independent of the Piwi protein. Finally, we present evidence that in all the tissues examined, the repression is controlled by the heterochromatic COM locus. CONCLUSION: Our data shed further light on the silencing mechanism that acts to target Drosophila LTR retrotransposons in somatic cells throughout fly development. They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter. They further demonstrate that these pathways are controlled by the heterochromatic COM locus which ensures the overall protection of Drosophila against the detrimental effects of random retrotransposon mobilization

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants

​The LInker of Nucleoskeleton and Cytoskeleton (LINC) complex is an evolutionary well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its implication in chromatin organisation has not been studied in plants. Here 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana, in which heterochromatin cluster in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences

Contrôle génétique par l’hôte de deux rétrovirus endogènes, ZAM et Idefix, chez Drosophila melanogaster.

Endogenous retroviruses colonize the genomes of all the vertebrates and are sometimes at the origin of pathologies. Few data are available on the relations which became between retroviruses and their host because the genetic studies to vertebrates are difficult to implement.ZAM and Idefix are two endogenous retrovirus of an invertebrate, Drosophila melanogaster, they were identified in our laboratory. They are mobilized in a genetically unstable lineage where their number of copies was amplified. We looked for the genetic determiners which control the mobilization of ZAM and Idefix in this lineage called Rev.We characterized ZAM element: ZAM has the genomic complete structure of an errantivirus, that is an endogenous retrovirus of insect. It expresses all the necessary composants for its cycle in the somatic cells of the adult ovaries of the lineage Rev, but in no other one of the lineages of drosophila which we tested. ZAM undergoes a lineage specific transcriptionnal control. We looked for the genetic determiners responsible for this control by means of transgenic drosophilas which possess a reporter gene drived by the LTR of ZAM. The LTR indeed contains sequences necessary for the expression of the element. We show that the LTR also possesses all the information to answer the lineage transcriptionnal control of and we localize the genetic determiners responsible for this control over the X chromosome of the host, in the cytogenetics site 20A. A parallel study on the characterization of Idefix led to the laboratory also highlighted a specific transcriptionnal control by the lineage. We show that determiners controlling Idefix have the same localization 20A. This region 20A is already known to control another errantivirus, gypsy. We thus called her "COM" for " Centre Organisateur de la Mobilisation ".Les rétrovirus endogènes colonisent les génomes de tous les vertébrés et sont parfois à l’origine de pathologies. Peu de données sont disponibles sur les relations qui se sont établies entre les rétrovirus et leur hôte car les études génétiques chez les vertébrés sont difficiles à mettre en oeuvre.ZAM et Idefix sont deux rétrovirus endogènes d’un invertébré, Drosophila melanogaster, qui ont été chacun identifiés dans notre laboratoire. Ils sont mobilisés dans une lignée génétiquement instable où leur nombre de copies a été amplifié. Nous avons recherché les déterminants génétiques qui contrôlent la mobilisation de ZAM et d’Idefix dans cette lignée appelée Rev.Nous avons d’abord caractérisé l’élément ZAM : ZAM possède la structure génomique complète d’un errantivirus, c’est à dire d’un rétrovirus endogène d’insecte. Il exprime tous les constituants nécessaires à son cycle dans les cellules somatiques des ovaires adultes de la lignée Rev, mais dans aucune autre des lignées de drosophile que nous avons testées. ZAM subit un contrôle transcriptionnel spécifique de souche. Nous avons recherché les déterminants génétiques responsables de ce contrôle à l’aide de drosophiles transgéniques qui possèdent un gène rapporteur dirigé par le LTR de ZAM. Le LTR contient en effet les séquences nécessaires à l’expression de l’élément. Nous montrons que le LTR possède également toute l’information pour répondre au contrôle transcriptionnel de souche et nous localisons les déterminants génétiques responsables de ce contrôle sur le chromosome X de l’hôte, au site cytogénétique 20A. Une étude parallèle sur la caractérisation d’Idefix menée au laboratoire a également mis en évidence un contrôle transcriptionnel spécifique de souche. Nous montrons que les déterminants contrôlant Idefix ont la même localisation 20A. Cette région 20A est déjà connue pour contrôler un autre errantivirus, gypsy. Nous l’avons donc appelée « COM » pour « Centre Organisateur de la Mobilisation »

CONTROLE GENETIQUE PAR L'HOTE DE DEUX RETROVIRUS ENDOGENES, ZAM ET IDEFIX, CHEZ DROSOPHILA MELANOGASTER (DOCTORAT)

CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Quantitative 3D Analysis of Nuclear Morphology and Heterochromatin Organization from Whole-Mount Plant Tissue Using NucleusJ.

International audienceImage analysis is a classical way to study nuclear organization. While nuclear organization used to be investigated by colorimetric or fluorescent labeling of DNA or specific nuclear compartments, new methods in microscopy imaging now enable qualitative and quantitative analyses of chromatin pattern, and nuclear size and shape. Several procedures have been developed to prepare samples in order to collect 3D images for the analysis of spatial chromatin organization, but only few preserve the positional information of the cell within its tissue context. Here, we describe a whole mount tissue preparation procedure coupled to DNA staining using the PicoGreen((R)) intercalating agent suitable for image analysis of the nucleus in living and fixed tissues. 3D Image analysis is then performed using NucleusJ, an open source ImageJ plugin, which allows for quantifying variations in nuclear morphology such as nuclear volume, sphericity, elongation, and flatness as well as in heterochromatin content and position in respect to the nuclear periphery

COM, a heterochromatic locus governing the control of independent endogenous retroviruses from Drosophila melanogaster.

ZAM and Idefix are two endogenous retroviruses whose expression is tightly controlled in Drosophila melanogaster. However, a line exists in which this control has been perturbed, resulting in a high mobilization rate for both retroviruses. This line is called the U (unstable) line as opposed to the other S (stable) lines. In the process of analyzing this control and tracing the genetic determinant involved, we found that ZAM and Idefix expression responded to two types of controls: one restricting their expression to specific somatic cells in the ovaries and the other silencing their expression in S lines but permitting it in U lines. While studying this second control in the U or S backgrounds, we found that the heterochromatic locus 20A2-3 on the X chromosome, previously implicated in the regulation of a third retroelement, gypsy, also controlled both ZAM and Idefix. We report here that genetic determinants necessary for endogenous retrovirus silencing occur at the 20A2-3 locus, which we call COM, for centre organisateur de mobilisation. We propose that if this point of control becomes mutated during the life of the fly, it may trigger processes reactivating dormant endogenous retroviruses and thus bring about sudden bursts of mobilization

Deep learning for nuclear image analysis and application to 3D plant nuclei

International audienceThe continuous development of microscopy has led biologists to have access to large high-resolution 2D and 3D image datasets. Automatic analysis of cellular and nuclear images has become an important challenge in the bioimaging field. To help biologists extract information from these images, tools have been designed to count objects in the image, study object type, their localization or morphology. The current state-of-the-art is led by deep learning methods. Their development relies on the availability of large data sets, on enhancing graphical processing units (GPU) of computers and on developments of new methodologies such as convolutional neural networks (CNN). However, non-IT users may experience difficulties when trying to use these on their own images. This short paper contains our first results after reviewing the state-of-the-art methods in the domain. It first introduces the current difficulties when working with bioimages, then lists the existing datasets for nuclear images analysis. It then exposes some of the easy-to-use tools for bioimaging and points out the different problems related to their use. It finally presents a new dataset for 3D images of plant nuclei that is designed for benchmarking purposes. This results should shortly be published in an journal of biology

Transcriptional properties and splicing of the flamenco piRNA cluster.

International audienceIn Drosophila, the piRNA cluster, flamenco, produces most of the piRNAs (PIWI-interacting RNAs) that silence transposable elements in the somatic follicle cells during oogenesis. These piRNAs are thought to be processed from a long single-stranded precursor transcript. Here, we demonstrate that flamenco transcription is initiated from an RNA polymerase II promoter containing an initiator motif (Inr) and downstream promoter element (DPE) and requires the transcription factor, Cubitus interruptus. We show that the flamenco precursor transcript undergoes differential alternative splicing to generate diverse RNA precursors that are processed to piRNAs. Our data reveal dynamic processing steps giving rise to piRNA cluster precursors

Probing the 3D architecture of the plant nucleus with microscopy approaches: challenges and solutions

The eukaryotic cell nucleus is a central organelle whose architecture determines genome function at multiple levels. Deciphering nuclear organizing principles influencing cellular responses and identity is a timely challenge. Despite many similarities between plant and animal nuclei, plant nuclei present intriguing specificities. Complementary to molecular and biochemical approaches, 3D microscopy is indispensable for resolving nuclear architecture. However, novel solutions are required for capturing cell-specific, sub-nuclear and dynamic processes. We provide a pointer for utilising high-to-super-resolution microscopy and image processing to probe plant nuclear architecture in 3D at the best possible spatial and temporal resolution and at quantitative and cell-specific levels. High-end imaging and image-processing solutions allow the community now to transcend conventional practices and benefit from continuously improving approaches. These promise to deliver a comprehensive, 3D view of plant nuclear architecture and to capture spatial dynamics of the nuclear compartment in relation to cellular states and responses